This invention relates to medicaments, particularly pyrimidine-5-carboxamide derivatives having Syk tyrosine kinase inhibition activity.
It is known that type I (immediate type) allergic reaction such as bronchial asthma, allergic rhinitis or atopic dermatitis is mediated mainly by the interaction between immunoglobulin E (IgE) and mast cells or basophiles. Mast cells and basophiles have an Fc receptor (Fcxcex5RI) having high affinity for IgE. Firstly, IgE binds to Fcxcex5RI and then antigens such as pollen, house dust or the like cross-link the receptor by binding to its specific IgE, thereby making progress of an allergic reaction. As a result of such a response, cytoplasmic secretory granules containing inflammatory mediators such as histamine, leukotriene and the like are released, which cause acute inflammatory reactions, and production of cytokine which takes part in various allergic and inflammatory reactions is accelerated.
It is known that at least two types of protein tyrosine kinase, Lyn (Eiseman, E. and Bolen, J. B., Nature, 355: 78-80 (1992)) and Syk (Taniguchi, T. et al., J. Biol. Chem., 266: 15790-15796 (1991)), are concerned in the intracellular signal transduction accompanied by this Fcxcex5RI activation. These tyrosine kinases are activated (tyrosine phosphorylated) after crosslinking of Fcxcex5RI by antigens (Hutchcroft, J. E. et al., Proc. Natl. Acad. Sci. USA, 89: 9107-9111 (1992)). It has been shown also that the SH2 domain and tyrosine kinase activity of Syk are necessary for the degranulation and cytokine production acceleration induced by the activation of Fcxcex5RI (Rivera, V. M. and Brugge, J. S., Mol. Cell. Biol., 15: 1582-1590 (1995)).
In consequence, it is expected that the release of mediators and the production of cytokines concerned in IgE stimulation-dependent allergic and inflammatory reactions from mast cells and basophiles can be controlled by inhibiting the tyrosine kinase activity of Syk.
It has been reported that the tyrosine phosphorylation of intracellular protein (activation) induced by stimulation of a receptor for IgG antibody, Fcxcex3R, and the phagocytosis mediated by Fcxcex3R are considerably inhibited in macrophages derived from Syk deficient mouse (Crowley, M. T. et al., J. Exp. Med., 186: 1027-1039 (1997)), suggesting that Syk has a markedly important role in the Fcxcex3R-mediated phagocytosis of macrophage. In consequence, there is a possibility that Syk inhibitors can inhibit cell or tissue damage induced by antibody-dependent cellular cytotoxicity (ADCC).
It has been reported that an antisense oligonucleotide of Syk suppresses the apoptosis inhibition of eosinophils induced by GM-CSF (Yousefi, S. et al., J. E. Med., 183: 1407-1414 (1996)), showing that Syk is essential for the life extending signal of eosinophils caused by GM-CSF and the like. Since life extension of eosinophils is closely related to the transition of diseases into a chronic state in allergic disorders such as asthma, Syk inhibitors can also become therapeutic agents for chronic eosinophilic inflammation.
Syk is important for the activation of B cells via a B cell antigen receptor and deeply concerned in the phosphatidylinositol metabolism and increase in the intracellular calcium concentration caused by the antigen receptor stimulation (Hutchcroft, J. E. et al., J. Biol. Chem., 267: 8613-8619 (1992) and Takata, M. et al., EMBO J., 13: 1341-1349 (1994)). In consequence, Syk inhibitors have a possibility of controlling the function of B cells and therefore are expected as therapeutic agents for antibody-related diseases.
Syk binds to a T cell antigen receptor, quickly undergoes tyrosine phosphorylation through crosslinking of the receptor and synergistically acts upon intracellular signals in which tyrosine kinases essential for the T cell activation such as Lck takes part (Couture, C. et al., Proc. Natl. Acad. Sci. USA, 91: 5301-5305 (1994) and Couture, C. et al., Mol. Cell. Biol., 14: 5249-5258 (1994)). In consequence, it is suggested that Syk inhibitors have a potential to be agents for inhibiting cellular immunity mediated by T cell antigen receptor.
Release of arachidonic acid and serotonin and platelet aggregation induced by collagen are markedly-inhibited in platelets derived from Syk deficient mouse (Poole, A. et al., EMBO J., 16: 2333-2341 (1997)), so that an anticoagulation action is also expected in Syk inhibitors.
On the other hand, WO 97/19065 discloses that a 2-anilinopyrimidine derivative represented by the following formula selectively inhibits p56lck, p59fyn, ZAP-70 and protein kinase C. However, it does not disclose or suggest about its action upon Syk. 
(In the formula, R6 represents H, xe2x80x94NH2, substituted amino, nitro, xe2x80x94COOH, ester or the like. See said document for other symbols.)
As pyrimidine compounds having substituted amino group at the 4-position and carboxamido group at the 5-position, the following compound 
is disclosed in Indian J. Chem., Sect. B, 16 (B) (10), 932-933 (1978), and the following compound 
is disclosed in EP 475206 and U.S. Pat. No. 5,104,877. However, there is no disclosure or suggestion about the action of these compounds upon Syk.
Also, antilipidemic activity of pyrimidine compounds having a phenylamino group at the 4-position is disclosed in EP 73328, U.S. Pat. No. 3,901,887, U.S. Pat. No. 3,910,910 and U.S. Pat. No. 3,940,394. However, these compounds do not have a carboxamido group at the 5-position, and there is no disclosure about the action upon Syk.
A plant natural product, Piceatannol, has so far been reported as a Syk tyrosine kinase inhibitor (Oliver, J. M. et al. , J. Biol. Chem., 269: 29697-29703 (1994)). However, since its in vitro Syk kinase inhibition activity is weak, great concern has been directed toward the creation of more excellent Syk tyrosine kinase inhibitor.
The present inventors have made extensive studies on compounds capable of inhibiting tyrosine kinase activity of Syk and, as a result, found that a pyrimidine derivative having a carboxamido group at the 5-position has excellent Syk tyrosine kinase inhibitory activity and a:Th is useful as an agent for preventing, treating or diagnosing diseases in which Syk takes part, and thereby have accomplished the invention.
Accordingly, the present invention relates to a pyrimidine-5-carboxamide derivative represented by the following general formula (I) or a salt thereof. 
[In the formula, each symbol has the following meaning;
X: O, S, NR1, CO, NR1CO, CONR1, Cxe2x95x90Nxe2x80x94OR1 or a bond,
Y: a lower alkylene group which may be substituted by OR1 or xe2x80x94NHR1, or a bond,
Z: O, NR2 or a bond,
A: H, or a lower alkyl which may have a substituent, a xe2x80x94CO-lower alkyl which may have a substituent, an aryl which may have a substituent, a heteroaryl which may have a substituent, a cycloalkyl which may have a substituent or a nitrogen-containing saturated heterocyclic group which may have a substituent,
B: an aryl which may have a substituent (provided that 2xe2x80x2-(1H-tetrazol-5-yl)biphenyl-4-yl group is excluded) or a heteroaryl group which may have a substituent, and
R1, R2: H, a lower alkyl or a xe2x80x94CO-lower alkyl group. The same shall apply hereinafter.]
Also, according to the present invention, there is provided a pharmaceutical composition, particularly a Syk tyrosine kinase inhibitor, which comprises the aforementioned pyrimidine-5-carboxamide derivative or a salt thereof.
The following further describes the compound of general formula (I).
In this specification, the term xe2x80x9clowerxe2x80x9d means a straight or branched hydrocarbon chain having from 1 to 6 carbon atoms. The xe2x80x9clower alkyl groupxe2x80x9d is preferably a lower alkyl group having from 1 to 4 carbon atoms, more preferably a methyl group, an ethyl group or an isopropyl group. The xe2x80x9clower alkylene groupxe2x80x9d is preferably a methylene group.
The xe2x80x9caryl groupxe2x80x9d is preferably a monocyclic to tricyclic aryl group having from 6 to 14 carbon atoms, more preferably, a phenyl group or a naphthyl group. Also, the phenyl group may be condensed with a five- to eight-membered cycloalkyl ring to form, for example, an indanyl group or a 5,6,7,8-tetrahydronaphthyl group, which is connected to the aromatic ring. The xe2x80x9ccycloalkyl groupxe2x80x9d is preferably a cycloalkyl group having from 3 to 8 carbon atoms, more preferably a cyclopropyl group, a cyclopentyl group or a cyclohexyl group. Also, the cycloalkyl group may be condensed with a benzene ring to form, for example, 1- or 2-indanyl or a 1,2,3,4-tetrahydronaphthyl group.
The xe2x80x9cnitrogen-containing saturated heterocyclic groupxe2x80x9d is a five- to eight-membered saturated heterocyclic group which has at least one N as a ring atom and may further have one O or S, and is preferably pyrrolidinyl, piperidyl, morpholinyl, piperazinyl, pyrazolidinyl, imidazolidinyl or homopiperazinyl. The xe2x80x9csaturated heterocyclic groupxe2x80x9d is a five- to eight-membered saturated heterocyclic group having from 1 to 4 hetero atoms selected from O, S and N, and it preferably includes tetrahydrofuranyl and tetrahydropyranyl, in addition to the groups described in the aforementioned xe2x80x9cnitrogen-containing saturated heterocyclic groupxe2x80x9d.
The xe2x80x9cheteroaryl group,xe2x80x9d is a five- to eight-membered monocyclic heteroaryl group having from 1 to 4 hetero atoms selected from 0, S and N, and it preferably includes pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl and thiazolyl groups.
Substituents of the xe2x80x9clower alkyl which may have a substituentxe2x80x9d, xe2x80x9caryl which may have a substituentxe2x80x9d, xe2x80x9cheteroaryl which may have a substituentxe2x80x9d, xe2x80x9ccycloalkyl which may have a substituentxe2x80x9d and xe2x80x9cnitrogen-containing saturated heterocyclic group which may have a substituentxe2x80x9d are not particularly limited so long as they can be used as substituents of these rings; but are preferably the substituents described in the following.
Substituents in the xe2x80x9ccycloalkyl which may have a substituentxe2x80x9d and xe2x80x9cnitrogen-containing saturated heterocyclic group which may have a substituentxe2x80x9d are preferably the groups selected from the following Group a, and they may have from 1 to 4 of these substituents. Particularly preferred are xe2x80x94NH2, xe2x80x94NH2 in a prodrug form and -lower alkylene-NH2.
Group a: xe2x80x94NH2, xe2x80x94NH2 in a prodrug form, -lower alkylene-NH2, -lower alkylene-NH2 in a prodrug form, xe2x80x94NH-lower alkyl, xe2x80x94N(lower alkyl)2, xe2x80x94NH-lower alkylene-aryl, xe2x80x94NH-aryl, xe2x80x94NH-cycloalkyl, xe2x80x94NH-heteroaryl, xe2x80x94NHCO-lower alkyl, xe2x80x94NHSO2-lower alkyl, xe2x80x94NHC(NH)NH2, xe2x80x94NHCONH2, xe2x80x94OH, xe2x80x94O-lower alkyl, xe2x80x94CO2H, xe2x80x94CONHOH, xe2x80x94CO2-lower alkyl, xe2x80x94CONH-lower alkyl and xe2x80x94CON(lower alkyl)2.
Substituents of the xe2x80x9caryl which may have a substituentxe2x80x9d, xe2x80x9cheteroaryl which may have a substituentxe2x80x9d are preferably the groups selected from the aforementioned Group a and the following Group b, and they may have from 1 to 4 of these substituents. Particularly preferred are xe2x80x94NH2, -lower alkylene-NH2, -lower alkyl, -halogen atom (F, Cl, Br or I), xe2x80x94CF3 and xe2x80x94O-lower alkyl group.
Group b: -lower alkyl, -halogen atom (F, Cl, Br or I), -lower alkyl substituted by a halogen atom (xe2x80x94CH2F, xe2x80x94CHF2, xe2x80x94CF3 or the like), xe2x80x94O-lower alkylene-aryl, xe2x80x94O-aryl, xe2x80x94O-lower alkylene-aryl-O-lower alkyl, xe2x80x94S-lower alkylene-aryl, xe2x80x94S-lower alkylene-aryl-O-lower alkyl, xe2x80x94NO2 and xe2x80x94CN.
The substituent of the xe2x80x9clower alkyl group which may have a substituentxe2x80x9d is preferably a group selected from the aforementioned Group a and the following Group c, and it may have from 1 to 4 of these substituents. Particularly preferred are xe2x80x94NH2 and xe2x80x94NH2 in a prodrug form.
Group c: -halogen atom (F, Cl, Br or I), xe2x80x94O-lower alkylene-aryl, xe2x80x94O-aryl, xe2x80x94O-lower alkylene-aryl-O-lower alkyl, xe2x80x94S-lower alkylene-aryl, xe2x80x94S-lower alkylene-aryl-O-lower alkyl, xe2x80x94NO2, xe2x80x94CN, -aryl which may be substituted by a group selected from the Group a, -cycloalkyl, -heteroaryl, -saturated hetero ring, -vinyl, -(1-propenyl) and -ethynyl.
Also, the xe2x80x9cxe2x80x94NH2 in a prodrug formxe2x80x9d means the groups well known by those skilled in the art, which becomes xe2x80x94NH2 under physiological conditions. Preferred are (Z)-3-[2-(acetoxy)phenyl]-2-propenoylamino-, (acetoxy)methoxycarbonylamino-, 4-azidobenzyloxycarbonylamino-, (5-methyl-2-oxo-1,3-dioxol-4-en-4-yl)methoxycarbonylamino- and [(2-hydroxyphenyl)(phenyl)methylidene]amino-, and other groups of this type known by those skilled in the art are also included.
In addition, the term xe2x80x9cbondxe2x80x9d means that the corresponding group does not exist, and the groups of both sides are directly linked.
Among compounds of the present invention, the following compounds can be cited as most preferred compounds: 2-(2-aminoethylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide, 2-(4-aminobutylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-bromoanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-nitroanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3,5-dimethylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(2-naphthylamino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3-bromoanilino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3,5-dichloroanilino)pyrimidine-5-carboxamide and 2-(cis-2-aminocyclohexylamino)-4-(3,4,5-trimethoxyanilino)pyrimidine-5-carboxamide.
Depending on the kinds of substituents, the compound of the present invention may exist in the form of geometrical isomers or tautomers, and isolated forms or mixtures of these isomers are included in the present invention. Also, the compound of the present invention may contain an asymmetric carbon atom in some cases, so that isomers based on the asymmetric carbon atom can exist. Mixtures or isolated forms of these optical isomers are included in the present invention.
Also, the compound of the present invention sometimes forms an acid addition salt or, depending on the kinds of substituent groups, a salt with a base. Such salts are pharmaceutically acceptable salts, and their illustrative examples include acid addition salts with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like) or with organic acids (e.g., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid and the like), salts with inorganic bases (e.g., sodium, potassium, magnesium, calcium, aluminum and the like) or with organic bases (e.g., methylamine, ethylamine, ethanolamine, lysine, ornithine and the like), ammonium salts, and the like. In addition, various types of hydrates and solvates and polymorphic substances of the compound (I) of the present invention and salts thereof are also included in the present invention.
The compound of the present invention and pharmaceutically acceptable salt thereof can be produced by applying various known synthesis methods, making use of L: their characteristics based on the basic structures or kinds of substituents. In this regard, depending on the kinds of functional groups, it is sometimes effective in view of the production techniques to replace said functional group by an appropriate protecting group, namely a group which can be easily converted into said functional group, at the step of the starting material or intermediate. Thereafter, the desired compound can be obtained by removing the protecting group as occasion demands. Examples of such functional groups include an amino group, a hydroxyl group, a carboxyl group and the like and examples of their protecting groups include the protecting groups described in xe2x80x9cProtective Groups in Organic Synthesis (2nd. Ed.)xe2x80x9d edited by Greene and Wuts, and these groups are optionally used depending on the reaction conditions.
The following describes typical production methods of the compound of the present invention.
Production method A 
(In the formulae, L represents a leaving group. The same shall apply hereinafter.)
This production method is a method in which the s:compound of the present invention represented by the general formula (I) is obtained by allowing a compound (II) to react with a compound (III). In this regard, examples of the leaving group L include halogen atoms and methylsulfanyl, 1H-benzotriazol-1-yloxy, methanesulfonyloxy, p-toluenesulfonyloxy, trifluoromethanesulfonyloxy and the like.
The reaction can be carried out from at room temperature to under heat reflux using the compounds (II) and (III) in equimolar amounts, or one of them in an excess amount, without solvent or in a solvent inert to the reaction such as aromatic hydrocarbons (e.g., benzene, toluene, xylene and the like), ethers (e.g., diethyl ether, tetrahydrofuran (THF), dioxane and the like), halogenated hydrocarbons (e.g., dichloromethane, 1,2-dichloroethane, chloroform and the like), and N,N-dimethylformamide (DMF), dimethyl acetamide (DMA), N-methylpyrrolidone, ethyl acetate, acetonitrile and the like. The reaction temperature can be optionally selected depending on the compounds. Depending on the compounds, it is advantageous in some cases to carry out the reaction in the presence of an organic base (preferably diisopropylethylamine, N-methylmorpholine, pyridine or 4-(N,N-dimethylamino)pyridine) or a metal salt base (preferably potassium carbonate or sodium hydroxide).
Production method B 
(Symbols in the formulae are as defined in the foregoing.)
This production method is a method in which the compound (I) of the present invention is obtained by converting the nitrile group of a nitrile compound (IV) into a carboxamido group under various conditions. The reaction can be carried out from at room temperature to under heat reflux without solvent or in an reaction inert solvent such as aromatic hydrocarbons, ethers, halogenated hydrocarbons, alcohols (e.g., methanol, ethanol and the like), DMF, pyridine, water, dimethyl sulfoxide (DMSO) and the like, in the presence of a mineral acid (e.g., sulfuric acid, hydrochloric acid, hydrobromic acid or the like), an organic acid (e.g., formic acid, acetic acid or the like), or a base (e.g., sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, ammonia or the like). It is advantageous in some cases to carry out the reaction in the presence of hydrogen peroxide or the like, in effecting smooth progress of the reaction. The reaction temperature can be selected optionally, depending on the compound.
Production method C 
(Symbols in the formulae are as defined in the foregoing.)
This production method is a method in which the compound (I) of the present invention is obtained by converting the carboxyl group of a compound (V) into carboxamido group under various conditions.
The production reaction can be carried out by treating the carboxylic acid compound (V) with ammonia without solvent or in a solvent inert to the reaction such as aromatic hydrocarbons, ethers, halogenated hydrocarbons, DMF, DMA, N-methylpyrrolidone, pyridine, DMSO, ethyl acetate, acetonitrile and the like, in the presence of a condensing agent (e.g., dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC), 1,1xe2x80x2-carbonylbis-1H-imidazole (CDI) or the like) and, in some cases, an additive agent such as N-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt) or the like. The reaction can be carried out from under cooling to under heat reflux and the reaction temperature can be selected optionally depending on the compound.
In this regard, when the compound (I) of the present invention in this production method has a hydroxyl group, an amino group and the like, these functional groups of the carboxylic acid compound (V) are protected in advance with a protecting group, and the protecting group is removed after completion of the reaction of the production method C. As the protecting group, it may be selected optionally from the protecting groups described in the aforementioned xe2x80x9cProtective Groups in Organic Synthesis (2nd. Ed.)xe2x80x9d.
Production method D 
(In the above formulae, R3 means a lower alkyl or R3xe2x80x94CONHxe2x80x94 as an amino group in a prodrug form, A1 taken together with NH2 or R3xe2x80x94CONHxe2x80x94 or a group derived from NH2 which will be described later represents A, and other symbols are as defined in the foregoing.)
This production method is a method in which a compound (Ib) of the present invention is obtained by allowing the amino group of a compound (Ia) having an amino group on A to react with a carboxylic acid compound.
In this reaction, condensation is effected for example by an acid halide method, a mixed or a symmetric acid anhydride method, an active ester method or a condensing agent (DCC, WSC, CDI or the like) method in an inert solvent such as halogenated hydrocarbons, ethers, DMF or the like from under cooling to under heating, preferably at from xe2x88x9220xc2x0 C. to 60xc2x0 C. It is advantageous in some cases to carry out the reaction in the presence of an organic base, from the viewpoint of effecting smooth progress of the reaction.
Production method E 
(In the formulae, R4 represents a lower alkyl, and other symbols are as defined in the foregoing.)
This production method is a method in which a compound (Ic) is obtained by carrying out sulfonylation of the amino group of the compound (Ia) having an amino group on A.
In this reaction, condensation of a sulfonyl chloride compound with the compound (Ia) is carried out in an inert solvent such as halogenated hydrocarbons, ethers, DMF or the like from under cooling to under heating, preferably at from xe2x88x9220xc2x0 C. to 60xc2x0 C. It is advantageous in some cases to carry out the reaction in the presence of an organic base, from the viewpoint of effecting smooth progress of the reaction.
Production method F 
(Symbols in the formulae are as defined in the foregoing.)
This production method is a method in which a compound (Id) of the present invention having a guanidino group is produced from the compound (Ia) having an amino group on A.
The reaction is carried out using the amine compound and an guanidino adding agent such as 3,5-dimethylpyrazole-1-carboxamidine nitrate, cyanamide, isothiourea derivatives, isourea derivatives or the like without solvent or in a solvent inert to the reaction such as aromatic hydrocarbons, ethers, halogenated hydrocarbons, alcohols, water, DMF, DMA, N-methylpyrrolidone, pyridine, DMSO, ethyl acetate, acetonitrile and the like. It is advantageous in some cases to carry out the reaction in the presence of an organic base or a metal salt base, from the viewpoint of effecting smooth progress of the reaction. These solvents may be used alone or as a mixture of two or more. The reaction can be carried out from under cooling to under heat reflux. The reaction temperature can be selected optionally depending on the compounds.
Production method G 
(Symbols in the formulae are as defined in the foregoing.)
This production method is a method in which a compound (Ie) having a urea group is produced from the compound (Ia) having an amino group on A.
This reaction is carried out using the amine compound, a urea adding agent such as a cyanic acid derivative (e.g., sodium cyanate, potassium cyanate or the like), isocyanate derivative, urea, cyanogen bromide or the like, without solvent or in a solvent inert to the reaction such as aromatic hydrocarbons, ethers, halogenated hydrocarbons, alcohols, water, DMF, DMA, N-methylpyrrolidone, pyridine, DMSO, ethyl acetate, acetonitrile and the like. It is advantageous in some cases to carry out the reaction in the presence of an acid such as acetic acid, hydrochloric acid or the like or an base such as sodium hydroxide, potassium hydroxide, from the viewpoint of effecting smooth progress of the reaction. These solvents may be used alone or as a mixture of two or more. The reaction can be carried out from under cooling to under heat reflux. The reaction temperature can be selected optionally depending on the compounds.
Production method H 
(In the formulae, R5 represents a resin for solid phase synthesis, and other symbols are as described in the foregoing.)
This production method is a solid phase synthesis method which comprises the following three steps.
(1) Fix to a Resin By Amidation
A compound (VII) can be obtained by effecting condensation of a carboxylic acid compound (VI) with an amino terminal-containing resin for solid phase synthesis (e.g., amino(methyl) resin, Rink amide resin or the like) in the same manner as the case of production method C.
(2) Introduction of Substituent Group
A compound (VIII) can be produced in the same manner as described in the production method A.
(3) Removal of Resin
The compound (I) of the present invention is produced by eliminating the resin from the compound (VIII). The reaction is carried out by treating it with a mineral acid or organic acid without solvent or in a solvent inert to the reaction such as aromatic hydrocarbons, ethers, halogenated hydrocarbons, alcohols, DMF, DMA, N-methylpyrrolidone, pyridine, DMSO, ethyl acetate, acetonitrile and the like. It is advantageous in some cases to carry out the reaction in the presence of an additive agent such as difluoroethanol, triethylsilane, triisopropylsilane, (thio)anisole or the like.
Production Method of Starting Compounds
Starting compounds for the compound of the present invention can be produced in the usual way, for example, by applying known reactions shown in the following synthetic pathway diagram.
Production method 1
(In the formulae, R6 represents an ester or nitrile group and Lxe2x80x2 represents a leaving group. The same shall apply hereinafter.)
The substitution reaction can be carried out in the same manner as the case of the aforementioned production method A. It is desirable to carry out the reaction in a solvent such as acetonitrile or the like in the presence of an organic base when X is NR2, or in a solvent such as DMF or the like in the presence of a metal salt base when X is O or S. Regarding the hydrolysis, when R6 is nitrile group, the amide compound (II) can be obtained by carrying out the same procedure of the aforementioned production method B and, when R6 is an ester group, the carboxylic acid compound (VI) can be obtained by treating with an acid or alkali in the usual way. The amidation can be carried out in the same manner as the case of the aforementioned production method C. In carrying out the amidation, the leaving group L may be substituted by other group (benzotriazol-1-yloxy, imidazolyl, amino or the like) depending on the conditions.
Production method 2
(In the formulae, R7 represents a lower alkyl or methylsulfanyl group, and other symbols are as defined in the foregoing.)
This production method is a method in which a pyrimidine ring is formed by allowing an alkoxymethylene compound (XI) to react with an amidine or isothiourea compound (XII) under a neutral or basic condition. Water, methanol, ethanol, 1,4-dioxane, pyridine or the like can be used as the solvent. These solvents may be used alone or as a mixture of two or more. Examples of the base to be used include potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, sodium ethoxide and the like. The reaction can be carried out generally from under room temperature to under heat refux. The reaction temperature can be optionally selected depending on the compounds.
The reaction product obtained by each of the aforementioned production methods is isolated and purified as a free compound, a salt thereof or various types of solvate such as hydrate. Salts can be produced by usual salt formation treatment.
The isolation and purification are carried out by employing usual chemical operations such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various chromatographic techniques and the like.
Various isomers can be isolated in the usual way making use of a physicochemibal difference among isomers. For example, optical isomers can be separated by a general optical resolution method such as fractional crystallization or a chromatography. I n addition, an optical isomer can also be produced from an appropriate optically active material compound.
The compound of the invention is useful as an active ingredient for pharmaceutical preparations. Particularly, since it has the action to inhibit Syk tyrosine kinase activity, it is useful as agents for preventing and treating diseases in which Syk takes part, including those diseases in which an allergic or inflammatory reaction becomes the main cause, such as allergic diseases (e.g., asthma, rhinitis, atopic dermatitis, contact dermatitis, nettle rash, food allergy, conjunctivitis, vernal conjunctivitis and the like), autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, psoriasis and the like, ulcerative diseases (e.g., ulcerative colitis and the like), fibrosing diseases, osteoarthritis, cancers and the like; diseases in which immune reaction takes part, such as rejection at the time of organ transplantation and graft versus host disease and the like; diseases in which ADCC takes part, such as autoimmune hemolytic anemia, myasthenia gravis and the like; and diseases in which platelet agglutination takes part, such as thrombosis and the like.
Actions of the compound of the invention have been confirmed by the following pharmacological tests.
1) Preparation of Syk Protein:
Human Syk gene, in which a gene of FLAG tag consisting of 8 amino acid residues was linked to the 3xe2x80x2-end, was cloned using RT-PCR method from total RNA prepared from Jurkat cells. The amplified cDNA was incorporated into a vector, pFASTBAC HT, contained in Baculovirus Expression System (GIBCO BRL Inc.). The pFASTBAC HT is designed in such a manner that a His tag consisting of 6 histidine residues can be fused to the 5xe2x80x2-end of Syk. This plasmid DNA was introduced into competent cells, DH10BAC, contained in the Baculovirus Expression System to prepare DNA of recombinant virus. Thereafter, the recombinant virus (culture supernatant) was obtained by transfection of the DNA of recombinant virus into Sf-9 cells (ATCC).
The Sf-9 cells infected with this recombinant virus were recovered and lysed using a lysis buffer containing 1% Triton X-100. After centrifugation of the soluble fraction, TALON resin (CLONTECH) was added to the supernatant to allow the His-tag fused Syk protein to be adsorbed by the resin. After several times of washing of the resin, the His-tag fused Syk protein was eluted with a E buffer containing imidazole.
2) Preparation of Band 3 Peptide:
A peptide of 18 amino acid residues (MEELQDDYEDMMEENLEQ) containing Tyr-8 of human erythrocyte Band 3 (Harrison, M. L. et al., J. Biol. Chem., 269: 955-959 (1994)) was synthesized using a peptide synthesizer. Using a biotinylation kit manufactured by Pierce, the N-terminal of the peptide under a resin-linked condition was biotinylated, and purification was carried out using an HPLC.
3) Measurement of Syk Tyrosine Kinase Activity Using an SPA System:
SPA (Scintillation Proximity Assay) is a system developed by Amersham making use of a phenomenon in which scintillation occurs when a molecule having a radioactivity is in the proximity of (linked to) the surface of plastic beads having a scintillant included therein. These beads are coated in advance with streptoavidin to which the biotin moiety of substrate peptide is bound.
A 2 xcexcl portion of DMSO solution of each compound to be tested (final DMSO concentration, 4%) was added to each well containing 50 xcexcl of a reaction solution (composition: 0.2 xcexcg Syk, 50 mM Tris-HCl (pH 8), 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.4 xcexcM Band 3 peptide and 0.1 xcexcCi [xcex3-33P]ATP (10 mCi/ml, Amersham)). This was prepared in OptiPlate(trademark) (PACKARD) and allowed to stand at room temperature (20 to 25xc2x0 C.) for 1 hour to effect tyrosine phosphorylation.
The reaction was terminated by adding PBS containing 0.25 mg SPA beads, 50 xcexcM ATP, 5 mM EDTA and 1% Triton X-100 (reaction termination solution) in an amount of 150 xcexcl per well.
The plate was sealed, stirred, allowed to stand at room temperature for 15 minutes and then centrifuged at 1,500 rpm for 3 minutes to effect precipitation of the SPA beads. Radioactivity of each well was measured using TOP COUNT (PACKARD), and the tyrosine phosphorylation activity by Syk was calculated.
4) Results:
The following compounds of the present invention showed an inhibition activity of 0.1 xcexcM or less as IC50 value upon Syk tyrosine kinase. 2-(2-Aminoethylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide, 2-(4-aminobutylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-bromoanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-nitroanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3,5-dimethylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(2-naphthylamino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3-bromoanilino)pyriznidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3,5-dichloroanilino)pyrimidine-5-carboxamide and 2-(cis-2-aminocyclohexylamino)-4-(3,4,5-trimethoxyanilino)pyrimidine-5-carboxamide.
This was carried out in accordance with the method reported by Collado-Escobar et al. (Collado-Escobar, D et al. J. Immunol., 144: 3449-3457 (1990)).
The compound of the present invention excellently inhibited release of 5-HT.
Male ICR (CD-1) mice of 5 weeks age were sensitized by subcutaneously injecting 10 xcexcl of anti-dinitrophenyl-IgE (DNP-IgE) (1,000 times dilution of a partially purified preparation of ascites of Balb/c mouse to which a DNP-IgE producing hybridoma had been administered by intraperitoneal injection) under the right ear pinna while w lightly anesthetizing with ether. After 24 hours of the sensitization, 200 xcexcl of 0.5% Evans blue solution containing 50 xcexcg of DNP-conjugated bovine serum albumin was intravenously administered, and then each mouse was sacrificed by exsanguination 30 minutes thereafter to isolate both ears. Each test compound or the vehicle alone as a control was subcutaneously administered 30 minutes before the antigen challenge. The dye in the tissues was extracted with formamide and colorimetrically determined at 620 nm. A value obtained by subtracting the dye content of the left ear from the dye content of the right ear was used as the amount of dye leaked into the tissues by the PCA reaction.
The PCA inhibition ratio by the test compound was calculated based on the following equation. In the formula, CA: amount of the dye leaked into the sensitized right ear at the time of the administration of the vehicle alone, CB: amount of the dye leaked into the un-sensitized left ear at the time of the administration of the vehicle alone, XA: amount of the dye leaked into the sensitized right ear at the time of the administration of the compound to be tested, and XB: amount of the dye leaked into the un-sensitized left ear at the time of the administration of the compound to be tested. Inhibition ratio (%)={(CAxe2x88x92CB)xe2x88x92(XAxe2x88x92XB)}xc3x97100/(CAxe2x88x92CB)
The compounds of the present invention excellently suppressed PCA reaction.
The pharmaceutical composition which contains one or two or more of the compounds represented by the general formula (I) or salts thereof as the active ingredient can be prepared by generally used methods using pharmaceutical carriers, fillers and the like which are generally used in this field. Its administration form may be either oral administration by tablets, pills, capsules, granules, powders, liquids and the like, or parenteral administration by intravenous, intramuscular and the like injections, suppositories, eye drops, eye ointments, percutaneous liquids, ointments, percutaneous adhesive preparations, transmucosal liquids, transmucosal adhesive preparations, inhalations and the like.
The solid composition for use in the oral administration according to the invention is used in the form of tablets, powders, granules and the like. In such a solid composition, one or more active substances are mixed with at least one inert diluent such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, aluminum magnesium silicate. In the usual way, the composition may contain other additives than the inert diluent, such as a lubricant (e.g., magnesium stearate or the like), a disintegrating agent (e.g., calcium cellulose glycolate or the like), a stabilizing agent (e.g., lactose or the like) and a solubilization assisting agent (e.g., glutamic acid, aspartic acid or the like). If necessary, tablets or pills may be coated with a film of a sugar coating, gastric or enteric substance such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate or the like.
The liquid composition for oral administration use includes pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like and contains a generally used inert diluent such as purified water or ethanol. In addition to the inert diluent, this composition may also contain auxiliary agents such as a solubilizing agent, a moistening agent, a suspending agent and the like, as well as sweeteners, flavors, aromatics and antiseptics.
The injections for parenteral administration use include aseptic aqueous or non-aqueous solutions, suspensions and emulsions. Examples of the diluent for use in the aqueous solutions and suspensions include distilled water for injection use and physiological saline. Examples of the diluent for use in the non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, plant oil such as olive oil, alcohols such as ethanol, polysorbate 80 (trade name) and the like. Such a composition may further contain auxiliary agents such as a tonicity agent, an antiseptic, a moistening agent, an emulsifying agent, a dispersing agent, a stabilizing agent (e.g., lactose) and a solubilization assisting agent (e.g., glutamic acid or aspartic acid). These compositions are sterilized by filtration through a bacteria retaining filter, blending of a germicide or irradiation. Alternatively, they may be used by firstly making into sterile solid compositions and dissolving them in sterile water or a sterile solvent for injection use prior to their use.
The transmucosal preparations such as transnasal preparations are in the solid, liquid or semisolid form and can be produced by known methods. For example, they are formed into a solid, liquid or semisolid state by optionally adding known pH adjusting agents, antiseptics, thickeners, excipients and the like. The transnasal preparations are administered using generally used sprayers, nasal drops containers, tubes, nasal cavity insertion tools and the like.
In the case of oral administration, the suitable daily dose is generally from about 0.001 to 100 mg/kg body weight, preferably from 0.1 to 10 mg/kg, which is administered in one portion or by dividing into two to four doses. In the case of intravenous injection, the suitable daily dose is from about 0.0001 to 10 mg/kg body weight, which is administered in one portion or by dividing into several doses. In the case of transmucosal preparations, a dose of from about 0.001 to 10 mg/kg body weight is administered once a day or by dividing into several doses. The dose is optionally decided by taking into consideration symptoms, age, sex and the like of each patient.